RESUMO
Chronic inflammation is implicated in many types of diseases, including cardiovascular, neurodegenerative, metabolic, and immune disorders. The search for therapeutic targets to control chronic inflammation often involves narrowing down the various molecules associated with pathology that have been discovered by various omics analyses. Herein, a different approach to identify therapeutic targets against chronic inflammation is proposed and one such target is discussed as an example. In chronically inflamed tissues, a large number of cells receive diverse proinflammatory signals, the intracellular signals are intricately integrated, and complicated intercellular interactions are orchestrated. This review focuses on effectively blocking this chaotic inflammatory signaling network via the endolysosomal system, which acts as a cellular signaling hub. In endolysosomes, the inflammatory signals mediated by pathogen sensors, such as Toll-like receptors, and the signals from nutrient and metabolic pathways are integrally regulated. Disruption of endolysosome signaling results in a strong anti-inflammatory effect by disrupting various signaling pathways, including pathogen sensor-mediated signals, in multiple immune cells. The endolysosome-resident amino acid transporter, solute carrier family 15 member 4 (SLC15A4), which plays an important role in the regulation of endolysosome-mediated signals, is a promising therapeutic target for several inflammatory diseases, including autoimmune diseases. The mechanisms by which SLC15A4 regulates inflammatory responses may provide a proof of concept for the efficacy of therapeutic strategies targeting immune cell endolysosomes.
RESUMO
The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.
Assuntos
Regulação da Expressão Gênica/fisiologia , Macrófagos/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células Dendríticas/metabolismo , Desoxiglucose/análogos & derivados , Desoxiglucose/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Macrófagos/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Oligodesoxirribonucleotídeos/farmacologiaRESUMO
SLC15A4 is a lysosome-resident, proton-coupled amino-acid transporter that moves histidine and oligopeptides from inside the lysosome to the cytosol of eukaryotic cells. SLC15A4 is required for Toll-like receptor 7 (TLR7)- and TLR9-mediated type I interferon (IFN-I) productions in plasmacytoid dendritic cells (pDCs) and is involved in the pathogenesis of certain diseases including lupus-like autoimmunity. How SLC15A4 contributes to diseases is largely unknown. Here we have shown that B cell SLC15A4 was crucial for TLR7-triggered IFN-I and autoantibody productions in a mouse lupus model. SLC15A4 loss disturbed the endolysosomal pH regulation and probably the v-ATPase integrity, and these changes were associated with disruption of the mTOR pathway, leading to failure of the IFN regulatory factor 7 (IRF7)-IFN-I regulatory circuit. Importantly, SLC15A4's transporter activity was necessary for the TLR-triggered cytokine production. Our findings revealed that SLC15A4-mediated optimization of the endolysosomal state is integral to a TLR7-triggered, mTOR-dependent IRF7-IFN-I circuit that leads to autoantibody production.
Assuntos
Formação de Anticorpos/imunologia , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas de Membrana Transportadoras/imunologia , Serina-Treonina Quinases TOR/imunologia , Animais , Anticorpos/imunologia , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Células Cultivadas , Imunoglobulina G/biossíntese , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/biossíntese , Lúpus Eritematoso Sistêmico/patologia , Lisossomos/fisiologia , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/imunologiaRESUMO
Thymic dendritic cells (DCs) promote immune tolerance by regulating negative selection of autoreactive T cells in the thymus. How DC homing to the thymus is transcriptionally regulated is still unclear. Microphthalmia-associated transcription factor (Mitf) is broadly expressed and plays essential roles in the hematopoietic system. Here, we used Mitf-mutated mice (Mitfvit/vit) and found enlargement of the thymus and expansion of CD4/CD8 double-positive T cells. Mitf was highly expressed in a subset of thymic DCs among the hematopoietic system. Genetic mutation or pharmacological inhibition of Mitf in DCs decreased the expression levels of Itga4, which are critical molecules for the homing of DCs to the thymus. Further, inhibition of Mitf decreased thymic DC number. These results suggest a pivotal role of Mitf in the maintenance of T cell differentiation by regulating the homing of DC subsets within the thymus.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Fator de Transcrição Associado à Microftalmia/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Células Dendríticas/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Hiperplasia , Integrina alfa4/genética , Integrina alfa4/imunologia , Integrina alfa4/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Timo/metabolismo , Timo/patologiaRESUMO
Controlling inflammation can alleviate immune-mediated, lifestyle-related and neurodegenerative diseases. The endolysosome system plays critical roles in inflammatory responses. Endolysosomes function as signal transduction hubs to convert various environmental danger signals into gene expression, enabling metabolic adaptation of immune cells and efficient orchestration of inflammation. Solute carrier family 15 member A3 (SLC15A3) and member A4 (SLC15A4) are endolysosome-resident amino acid transporters that are preferentially expressed in immune cells. These transporters play essential roles in signal transduction through endolysosomes, and the loss of either transporter can alleviate multiple inflammatory diseases because of perturbed endolysosome-dependent signaling events, including inflammatory and metabolic signaling. Here, we summarize the findings leading to a proof-of-concept for anti-inflammatory strategies based on targeting SLC15 transporters.
Assuntos
Sistemas de Transporte de Aminoácidos/imunologia , Inflamação/imunologia , Animais , Humanos , Lisossomos/imunologiaRESUMO
Solute carrier family 15 member 4 (SLC15A4) is an endolysosome-resident amino acid transporter that regulates innate immune responses, and is genetically associated with inflammatory diseases such as systemic lupus erythematosus (SLE) and colitis. SLC15A4-deficient mice showed the amelioration of symptoms of these model diseases, and thus SLC15A4 is a promising therapeutic target of SLE and colitis. For developing a SLC15A4-based therapeutic strategy, understanding human SLC15A4's properties is essential. Here, we characterized human SLC15A4 and demonstrated that human SLC15A4 possessed pH- and temperature-dependent activity for the transportation of dipeptides or tripeptides. Human SLC15A4 localized in LAMP1+ compartments and constitutively associated with Raptor and LAMTORs. We also investigated SLC15A4's role in inflammatory responses using the human plasmacytoid dendritic cell line, CAL-1. Knock down (KD) of the SLC15A4 gene in CAL-1 (SLC15A4-KD CAL-1) impaired Toll-like receptor (TLR) 7/8 or TLR9-triggered type I interferon (IFN-I) production and mTORC1 activity, indicating that human SLC15A4 is critical for TLR7/8/9-mediated inflammatory signaling. We also examined SLC15A4's role in the autophagy response since SLC15A4 loss caused the decrease of mTORC1 activity, which greatly influences autophagy. We found that SLC15A4 was not required for autophagy induction, but was critical for autophagy sustainability. Notably, SLC15A4-KD CAL-1 severely decreased mitochondrial membrane potential in starvation conditions. Our findings revealed that SLC15A4 plays a key role in mitochondrial integrity in human cells, which might benefit immune cells in fulfilling their functions in an inflammatory milieu.
Assuntos
Interferon Tipo I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Toll-Like/metabolismo , Animais , Linhagem Celular , Colite/metabolismo , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Inflamação/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Macrophage and dendritic cell (DC) progenitors (MDPs) and common DC progenitors (CDPs) are bone marrow (BM) progenitors with DC differentiation potential. However, both MDPs and CDPs give rise to large numbers of conventional DCs (cDCs) and few plasmacytoid DCs (pDCs), implying that more dedicated pDC progenitors remain to be identified. Here we have described DC progenitors with a prominent pDC differentiation potential. Although both MDPs and CDPs express the macrophage colony stimulating factor (M-CSF) receptor (M-CSFR), the progenitors were confined to a M-CSFR(-) fraction, identified as Lin(-)c-Kit(int/lo)Flt3(+)M-CSFR(-), and expressed high amounts of E2-2 (also known as Tcf4) an essential transcription factor for pDC development. Importantly, they appeared to be directly derived from either CDPs or lymphoid-primed multipotent progenitors (LMPPs). Collectively, our findings provide insight into DC differentiation pathways and may lead to progenitor-based therapeutic applications for infection and autoimmune disease.
Assuntos
Células Dendríticas/metabolismo , Células Progenitoras Linfoides/metabolismo , Linfopoese/imunologia , Macrófagos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/biossíntese , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células Progenitoras Linfoides/imunologia , Linfopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/deficiência , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator de Transcrição 4 , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Type I interferon (IFN-I) is a family of multifunctional cytokines that modulate the innate and adaptive immunity and are used to treat mastocytosis. Although IFN-I is known to suppress mast cell function, including histamine release, the mechanisms behind its effects on mast cells have been poorly understood. We here investigated IFN-I's action on mast cells using interferon-α/ß receptor subunit 1 (Ifnar1)-deficient mice, which lack a functional IFN-I receptor complex, and revealed that IFN-I in the steady state is critical for mast cell homeostasis, the disruption of which is centrally involved in systemic anaphylaxis. Ifnar1-deficient mice showed exacerbated systemic anaphylaxis after sensitization, which was associated with increased histamine in the circulation, even though the mast cell numbers and high affinity immunoglobulin E receptor (FcεRI) expression levels were similar between Ifnar1-deficient and wild-type (WT) mice. Ifnar1-deficient mast cells showed increased secretory granule synthesis and exocytosis, which probably involved the increased transcription of Tfeb. Signal transducer and activator of transcription 1(Stat1) and Stat2 were unexpectedly insufficient to mediate these IFN-I functions, and instead, Stat3 played a critical role in a redundant manner with Stat1. Our findings revealed a novel regulation mechanism of mast cell homeostasis, in which IFN-I controls lysosome-related organelle biogenesis.
Assuntos
Anafilaxia/imunologia , Interferon Tipo I/fisiologia , Mastócitos/imunologia , Vesículas Secretórias/metabolismo , Animais , Células Cultivadas , Histamina/sangue , Homeostase , Camundongos , Receptor de Interferon alfa e beta/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/fisiologia , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/fisiologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/fisiologia , Transdução de SinaisRESUMO
BACKGROUND: Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. METHODS: Upstream signaling cascades mediated by several known receptors in bone marrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. RESULTS: All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1. CONCLUSIONS: Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose-dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag-, SCF-, or HSP70-stimulated mast cells occur at the level of Lck-LAT phosphorylation.
Assuntos
Degranulação Celular , Mastócitos , Animais , Ligantes , Mastócitos/metabolismo , Camundongos , Fosforilação , Receptores CCR1 , Receptores de IgE/metabolismo , Transdução de SinaisRESUMO
Neutrophils rapidly undergo polarization and directional movement to infiltrate the sites of infection and inflammation. Here, we show that an inhibitory MHC I receptor, Ly49Q, was crucial for the swift polarization of and tissue infiltration by neutrophils. During the steady state, Ly49Q inhibited neutrophil adhesion by preventing focal-complex formation, likely by inhibiting Src and PI3 kinases. However, in the presence of inflammatory stimuli, Ly49Q mediated rapid neutrophil polarization and tissue infiltration in an ITIM-domain-dependent manner. These opposite functions appeared to be mediated by distinct use of effector phosphatase SHP-1 and SHP-2. Ly49Q-dependent polarization and migration were affected by Ly49Q regulation of membrane raft functions. We propose that Ly49Q is pivotal in switching neutrophils to their polarized morphology and rapid migration upon inflammation, through its spatiotemporal regulation of membrane rafts and raft-associated signaling molecules.
Assuntos
Microdomínios da Membrana/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neutrófilos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Animais , Movimento Celular/genética , Movimento Celular/imunologia , Polaridade Celular/genética , Polaridade Celular/imunologia , Células Cultivadas , Adesões Focais/genética , Adesões Focais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Quinases da Família src/antagonistas & inibidoresRESUMO
Mast cells possess specialized lysosomes, so-called secretory granules, which play a key role not only in allergic responses but also in various immune disorders. The molecular mechanisms that control secretory-granule formation are not fully understood. Solute carrier family member 15A4 (SLC15A4) is a lysosome-resident amino-acid/oligopeptide transporter that is preferentially expressed in hematopoietic lineage cells. Here, we demonstrated that SLC15A4 is required for mast-cell secretory-granule homeostasis, and limits mast-cell functions and inflammatory responses by controlling the mTORC1-TFEB signaling axis. In mouse Slc15a4-/- mast cells, diminished mTORC1 activity increased the expression and nuclear translocation of TFEB, a transcription factor, which caused secretory granules to degranulate more potently. This alteration of TFEB function in mast cells strongly affected the FcεRI-mediated responses and IL-33-triggered inflammatory responses both in vitro and in vivo. Our results reveal a close relationship between SLC15A4 and secretory-granule biogenesis that is critical for the functional integrity of mast cells.
Assuntos
Inflamação/imunologia , Lisossomos/metabolismo , Mastócitos/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Vesículas Secretórias/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Degranulação Celular , Linhagem Celular , Homeostase , Interleucina-33/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Ratos , Receptores de IgG/metabolismo , Transdução de SinaisRESUMO
Specific HLA class II alleles are strongly associated with susceptibility to rheumatoid arthritis (RA); however, how HLA class II regulates susceptibility to RA has remained unclear. Recently, we found a unique function of HLA class II molecules: their ability to aberrantly transport cellular misfolded proteins to the cell surface without processing to peptides. Rheumatoid factor (RF) is an autoantibody that binds to denatured IgG or Fc fragments of IgG and is detected in 70-80% of RA patients but also in patients with other diseases. Here, we report that intact IgG heavy chain (IgGH) is transported to the cell surface by HLA class II via association with the peptide-binding groove and that IgGH/HLA class II complexes are specifically recognized by autoantibodies in RF-positive sera from RA patients. In contrast, autoantibodies in RF-positive sera from non-RA individuals did not bind to IgGH/HLA class II complexes. Of note, a strong correlation between autoantibody binding to IgG complexed with certain HLA-DR alleles and the odds ratio for that allele's association with RA was observed (r = 0.81; P = 4.6 × 10(-5)). Our findings suggest that IgGH complexed with certain HLA class II alleles is a target for autoantibodies in RA, which might explain why these HLA class II alleles confer susceptibility to RA.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Suscetibilidade a Doenças/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoglobulina G/imunologia , Artrite Reumatoide/etiologia , Primers do DNA/genética , DNA Complementar/genética , Citometria de Fluxo , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Razão de Chances , Plasmídeos/genética , Dobramento de ProteínaRESUMO
Plasmacytoid dendritic cells (pDC) are the major producers of type I IFN during the initial immune response to viral infection. Ly49Q, a C-type lectin-like receptor specific for MHC-I, possesses a cytoplasmic ITIM and is highly expressed on murine pDC. Using Ly49Q-deficient mice, we show that, regardless of strain background, this receptor is required for maximum IFN-α production by pDC. Furthermore, Ly49Q expression on pDC, but not myeloid dendritic cells, is necessary for optimal IL-12 secretion, MHC-II expression, activation of CD4(+) T cell proliferation, and nuclear translocation of the master IFN-α regulator IFN regulatory factor 7 in response to TLR9 agonists. In contrast, the absence of Ly49Q did not affect plasmacytoid dendritic cell-triggering receptor expressed on myeloid cells expression or pDC viability. Genetic complementation revealed that IFN-α production by pDC is dependent on an intact tyrosine residue in the Ly49Q cytoplasmic ITIM. However, pharmacological inhibitors and phosphatase-deficient mice indicate that Src homology 2 domain-containing phosphatase 1 (SHP)-1, SHP-2, and SHIP phosphatase activity is dispensable for this function. Finally, we observed that Ly49Q itself is downregulated on pDC in response to CpG exposure in an ITIM-independent manner. In conclusion, Ly49Q enhances TLR9-mediated signaling events, leading to IFN regulatory factor 7 nuclear translocation and expression of IFN-I genes in an ITIM-dependent manner that can proceed without the involvement of SHP-1, SHP-2, and SHIP.
Assuntos
Células Dendríticas/imunologia , Interferon-alfa/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/fisiologia , Animais , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Teste de Complementação Genética/métodos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/farmacologia , Estrutura Terciária de Proteína/genética , Transporte Proteico/genética , Transporte Proteico/imunologiaRESUMO
Plasmacytoid dendritic cells (pDCs), originating from hematopoietic progenitor cells in the BM, are a unique dendritic cell subset that can produce large amounts of type I IFNs by signaling through the nucleic acid-sensing TLR7 and TLR9 (TLR7/9). The molecular mechanisms for pDC function and development remain largely unknown. In the present study, we focused on an Ets family transcription factor, Spi-B, that is highly expressed in pDCs. Spi-B could transactivate the type I IFN promoters in synergy with IFN regulatory factor 7 (IRF-7), which is an essential transcription factor for TLR7/9-induced type I IFN production in pDCs. Spi-B-deficient pDCs and mice showed defects in TLR7/9-induced type I IFN production. Furthermore, in Spi-B-deficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this uneven distribution was likely because of defective retainment of mature nondividing pDCs in the BM. The expression pattern of cell-surface molecules in Spi-B-deficient mice indicated the involvement of Spi-B in pDC development. The developmental defects of pDCs in Spi-B-deficient mice were more prominent in the BM than in the peripheral lymphoid organs and were intrinsic to pDCs. We conclude that Spi-B plays critical roles in pDC function and development.
Assuntos
Células da Medula Óssea/metabolismo , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Proteínas Proto-Oncogênicas c-ets/genética , Animais , Sequência de Bases , Células da Medula Óssea/fisiologia , Células Dendríticas/fisiologia , Citometria de Fluxo , Células HEK293 , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Proto-Oncogênicas c-ets/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/fisiologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/fisiologia , Ativação TranscricionalRESUMO
RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.
Assuntos
Citosol , Proteínas de Drosophila , Endocitose , Lisossomos , Interferência de RNA , RNA de Cadeia Dupla , Animais , RNA de Cadeia Dupla/metabolismo , Lisossomos/metabolismo , Citosol/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Linhagem Celular , Membranas Intracelulares/metabolismo , Permeabilidade , Drosophila/metabolismo , Drosophila/genéticaRESUMO
Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in trans on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in cis, but the role of this interaction is uncertain. Ly49Q, an atypical Ly49 receptor expressed in non-NK cells, binds MHC-I in cis and mediates chemotaxis of neutrophils and type I interferon production by plasmacytoid dendritic cells. We identified a lipid-binding motif in the juxtamembrane region of Ly49Q and found that Ly49Q organized functional membrane domains comprising sphingolipids via sulfatide binding. Ly49Q recruited actin-remodeling molecules to an immunoreceptor tyrosine-based inhibitory motif, which enabled the sphingolipid-enriched membrane domain to mediate complicated actin remodeling at the lamellipodia and phagosome membranes during phagocytosis. Thus, Ly49Q facilitates integrative regulation of proteins and lipid species to construct a cell type-specific membrane platform. Other Ly49 members possess lipid binding motifs; therefore, membrane platform organization may be a primary role of some NK cell receptors.
Assuntos
Esfingolipídeos , Animais , Humanos , Esfingolipídeos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fagocitose , Fagócitos/imunologia , Fagócitos/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Membrana Celular/metabolismo , Ligação ProteicaRESUMO
Although oral tolerance is a critical system in regulating allergic disorders, the mechanisms by which dietary factors regulate the induction and maintenance of oral tolerance remain unclear. To address this, we explored the differentiation and function of various immune cells in the intestinal immune system under fasting and ad libitum-fed conditions before oral ovalbumin (OVA) administration. Fasting mitigated OVA-specific Treg expansion, which is essential for oral tolerance induction. This abnormality mainly resulted from functional defects in the CX3CR1+ cells responsible for the uptake of luminal OVA and reduction of tolerogenic CD103+ dendritic cells. Eventually, fasting impaired the preventive effect of oral OVA administration on asthma and allergic rhinitis development. Specific food ingredients, namely carbohydrates and arginine, were indispensable for oral tolerance induction by activating glycolysis and mTOR signaling. Overall, prior food intake and nutritional signals are critical for maintaining immune homeostasis by inducing tolerance to ingested food antigens.
Assuntos
Arginina , Células Dendríticas , Tolerância Imunológica , Ovalbumina , Linfócitos T Reguladores , Serina-Treonina Quinases TOR , Animais , Arginina/metabolismo , Linfócitos T Reguladores/imunologia , Ovalbumina/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Camundongos Endogâmicos C57BL , Administração Oral , Receptor 1 de Quimiocina CX3C/metabolismo , Intestinos/imunologia , Antígenos CD/metabolismo , Cadeias alfa de Integrinas/metabolismo , Açúcares/metabolismo , Glicólise , Jejum , Transdução de Sinais , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , FemininoRESUMO
Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8(-/-) and Capn9(-/-) mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8(-/-) stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9(-/-). Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed "G-calpain," in which both were essential for activity. This is the first example of a "hybrid" calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S "knock-in" (Capn8(CS/CS)) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8(-/-) stomach, that of the Capn8(CS/CS) mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.